【作者】 陈倩；

【导师】 孙兴怀； 沈颖； 宋月莲；

【作者基本信息】 复旦大学 ， 眼科学， 2006， 博士

【摘要】 视神经损伤后初级视皮层神经元是否发生不可逆性变性或死亡是神经保护及人工视觉相关课题中的研究热点。本课题拟从实验角度出发探讨急慢性视神经损伤后视觉通路中初级视皮层的变化及其可能的机制。第一部分：大鼠急性视神经损伤后初级视皮层神经元的早期变化本部分的研究目的在于探讨急性视神经损伤早期即早基因c-jun、c-fos在初级视皮层的表达分布及其与视神经损伤程度的关系；此外了解视神经损伤早期初级视皮层神经元总数以及接受LGN输入的Ⅳ层神经元的数量是否发生改变。采用视神经钳夹和视神经离断的方法分别造成SD大鼠单眼急性部分视神经损伤和视神经完全性损伤的动物模型。分别于正常组和损伤后2h、1d、3d、1w、1m取材行冰冻切片，以免疫荧光组织化学染色法检测大鼠损伤眼代表区初级视皮层的c-Jun、c-Fos蛋白表达并计算阳性神经元分布密度，此外检测NeuN标记的初级视皮层神经元密度及接受LGN输入的Ⅳ层神经元密度，采用方差分析方法进行统计学检验。结果显示视神经损伤后2h初级视皮层即出现c-Jun蛋白的表达升高，1d达高峰；此后降低，但至1m时达另一峰值。视神经离断组c-Jun升高幅度高于视神经钳夹组，且高峰期维持时间较久。视皮层的c-Fos蛋白表达在视神经钳夹伤后2h降低，3d达谷底，之后回升；视神经离断组降低幅度小于视神经钳夹组，且2h时存在短暂表达升高。急性视神经损伤早期NeuN标记的初级视皮层的神经元密度和Ⅳ层神经元密度与正常组相比均无明显改变。第一部分的研究结果表明大鼠急性视神经损伤后2h初级视皮层即发生即早基因c-jun、c-fos的表达改变。急性视神经损伤早期(尤其2h～3d)初级视皮层的c-Jun、c-Fos蛋白表达呈相反态势，提示这两组转录因子可能通过作用相反的途径在初级视皮层引起不同的效应。除损伤程度的影响以外，c-Jun和c-Fos蛋白之间可能还存在某种平衡机制参与调节这两种转录因子的表达水平。第二部分：猕猴慢性高眼压模型初级视皮层的神经元改变第二部分的研究目的在于探讨不同时期猕猴慢性高眼压模型初级视皮层神经元的代谢和神经化学改变及其与RGC损失的关系，并了解不同时期猕猴慢性高眼压模型的初级视皮层神经元数量以及超微结构是否发生变化。研究对象为4只正常猕猴和11只通过激光小梁网烧灼方法造成的不同时期的慢性高眼压猕猴。取材于V1区视皮层，做冠状切面和切线位的脑组织冰冻切片，分别用组织化学方法进行细胞色素氧化酶(CO)的染色，荧光免疫组化方法进行NeuN神经元标记、GFAP胶质细胞活性检测、神经营养因子BDNF及其受体TrkB表达的检测。细胞色素氧化酶染色结果计算平均灰度值，NeuN标记的神经元计算其4C层的分布密度，GFAP和BDNF表达均计算阳性区域面积，分析结果与RGC轴突损失率之间的关系。并用透射电镜的方法观察初级视皮层4层神经元的超微结构改变。统计学分析采用方差分析法。结果显示正常组猕猴V1区视皮层4层CO染色均匀且致密，青光眼组猕猴因双眼分期不同4C层呈现深浅交替的眼优势柱结构，双眼绝对期者呈均匀一致的淡染。灰度值计算结果显示RGC轴突损失＜50％的模型眼代表区4C层平均灰度值与正常组相比无明显差异，而轴突损失＞50％的模型眼代表区4C层平均灰度值与正常组相比有高度统计学差异。4C层神经元计数在青光眼组与正常组间无统计学差异。与CO染色结果类似，当轴突损失＜50％时，模型眼代表区4C层GFAP表达与正常组无明显差异，当轴突损失＞50％时，模型眼代表区4C层GFAP表达明显高于正常组。青光眼组猕猴视皮层BDNF表达呈普遍性降低但分布均匀，且与RGC损失程度无关；TrkB表达也呈不同程度降低。青光眼组猕猴初级视皮层的超微结构改变包括尼氏体减少、线粒体肿胀、内质网扩张、脂褐素数量增加等，并无神经元不可逆变性或死亡的证据。以上结果表明在猕猴慢性高眼压模型中，初级视皮层表现出与RGC轴突损失率相关的神经元代谢活性改变和胶质细胞的活化，50％的RGC轴突损失率可能是视觉中枢皮层水平代谢活性改变的关键点，提示神经保护措施的实施可能在青光眼病程早中期RGC损失小于50％时更为有效。BDNF和TrkB的表达降低，表明初级视皮层神经元的功能抑制状态，并可能通过下行营养支持的削弱参与了青光眼病理机制中的恶性循环。鉴于在急慢性视神经损伤后初级视皮层均未发生神经元数量的减少，神经元超微结构也无神经元不可逆变性或死亡的证据，提示视神经损伤后的初级视皮层改变可能仅为皮层突触可塑性的一种表现形式。更多还原

【Abstract】 Whether transneuronal degeneration or neuronal death in the primary visual cortex happens after optic nerve injury has become a hot spot in the research field of neuroprotection and vision restoration. The present study was designed to investigate the characteristic changes and their potential mechanisms in primary visual cortex after acute or chronic optic nerve injuries.Part I: Early changes in the primary visual cortex of rats after acute optic nerve injuryIn the first part of the thesis, our research work was planed to investigate the differential expression of immediate-early genes c-jun and c-fos in primary visual cortex of rat early after acute optic nerve injury and the relationship between immediate-early gene expression and the injury degree of optic nerve. Furthermore, we wanted to find out if the total neuronal amount of primary visual cortex and neuronal amount of lamina IV changed early after acute optic nerve injury.Unilateral optic nerve crush or optic nerve transection was performed in Sprague-Dawley rats to obtain acute partial or complete monocular optic nerve injury models. Frozen sections through visual cortex were cut in normal rats and model rats respectively at 2h, 1d, 3d, 1w and 1m after operation. The differential expression of c-Jun and c-Fos was detected in the primary visual cortex of rats by means of immunohistochemistry. The number of NeuN labeled positive neurons in the arrowy sections of primary visual cortex and layer IV were calculated. Statistical comparisons were made using one-way ANOVA.The increased expression of c-Jun in the primary visual cortex could be observed at 2h postoperation, and reached peak value at 1d postoperation. Then it dropped and reached another peak value until lm postoperation. The extent of increase of c-Jun expression was much higher and the maintenance of fastigium was much longer in optic nerve transection models than in optic nerve crush models. The decreasedexpression of c-Fos in the primary visual cortex could be observed at 2h postoperation in optic nerve crush models, and reached valley value at 3d postoperation followed by increased expression. The extent of decrease of c-Fos expression was lower in optic nerve transection models than in optic nerve crush injury models and transient increased expression could be observed at 2h postoperation. Statistic analyses showed there were no significant changes exist between normal and model rats for total neuronal density in primary visual cortex or neuronal density in lamina IV.The results of part I indicated that the expression of immediate-early gene c-jun and c-fos changed shortly after optic nerve injury at 2h postoperation. The opposite expression of c-Jun and c-Fos in the primary visual cortex in early stage, especially 2h~3d, postoperation of optic nerve injury pointed out for us that these two groups of transcription factors may act in opposite direction in the primary visual cortex. Besides the direct influences of different injury degrees, some kind of equilibrium mechanisms may participate in accommodating the expression of these two transcription factors.Part II: Neuronal changes of primary visual cortex in chronic ocular hypertension models of monkeyPart II of this thesis was designed to investigate the metabolic and neurochemical changes in the primary visual cortex of different stages of monkey chronic ocular hypertension models along with their connection with RGC loss. Another objective of part II was to make it clear whether the amount and ultrastructure of neurons changed in the primary visual cortex of monkey glaucomatous models.The objects of our research work were 4 normal monkeys and 11 monkeys with unilateral or bilateral chronic ocular hypertension induced by laser scarification of the trabecular meshwork. Coronal and tractional sections of visual cortex were cut on a cryomicrotome. The cytochrome oxidase reaction, calculation of NeuN labeled neurons and distribution of three neurochemicals (GFAP, BDNF, TrkB) revealed by immunohistochemistry were studied. The results of cytochrome oxidase reaction were transformed into average greyness values. The spacial distribution of NeuN labeled neurons and positive area of GFAP or BDNF expression in lamina 4C of primary visual cortex were counted and the relationships between the results and RGC loss were analysed. Furthermore, the ultrastructural changes of neurons in lamina 4 of primary visual cortex were observed by means of transmission electron microscopy.Statistical comparisons were made using one-way ANOVA.Dense and continuous bands of high oxidative enzymatic activity were seen within lamina 4C of primary visual cortex in normal monkeys. A pattern of light bands alternating with dark bands in lamina 4C standing the structure of ocular dominance columns emerged in glaucomatous monkeys according to different stages of both eyes. While pale stained and continuous bands of low oxidative enzymatic activity could be seen within lamina 4C in binocular terminal-stage glaucomatous monkeys. Statistical analyses revealed there were no significant differences between the oxidative enzymatic activities in ocular dominance columns representing normal eyes and model eyes with RGC loss less than 50%. While in the ocular dominance columns representing model eyes with RGC loss more than 50%, the oxidative enzymatic activities were obviously under normal levels. There were no statistical differences of the neuronal amounts in lamina 4C between normal and glaucomatous monkeys. Similar to the results of cytochrome oxidase reaction, there were no significant differences of GFAP expression between ocular dominance columns representing normal eyes and model eyes with RGC loss less than 50%. While in the ocular dominance columns representing model eyes with RGC loss more than 50%, the GFAP expression were obviously above normal levels. The BDNF and TrkB expression was incoordinately decreased in glaucomatous monkeys, and independent of RGC loss. The ultrastructural changes of neurons in lamina 4 of glaucomatous monkeys included reduced quantity of ribosomes, swelled mitochondrias, dilated endoplasmic reticulums and increased amount of lipofuscin bodies. But there were no evidences indicated irreversible neuronal degeneration or apoptosis.These results related the neurochemical changes and glial activation in primary visual cortex to RGC loss in monkey ocular hypertension models. RGC loss of 50% might be a key-point for changes of metabolic activity at cortical level in visual centrum. With a view to this point, neuroprotection might be more effective while practiced in early to middle stage in the course of glaucoma with RGC loss less than 50%. The decreased expression of BDNF and TrkB indicated restrained neuronal activity in primary visual cortex, which might participate in the vicious circle in glaucomatous pathogenesis via lessened retrograde nutritional sustainments.Considering neither reduction of neuronal quantity in primary visual cortex nor evidences of irreversible degeneration of neuronal ultrastructure having been observed, neuronal changes of primary visual cortex in acute and chronic optic nerveinjury models were more likely representations of cortical plasticity.更多还原